Method of detecting genetic material in a biological sample and a device for its implementation

ABSTRACT

The object of the invention is a method of detecting genetic material in a biological sample in which the biological sample is loaded into the reaction cartridge ( 6 ) and then the reaction cartridge ( 6 ) is placed in the control device, the collected biological sample is taken to the isolation chamber ( 7 ), isolation of biological material from the tested sample by heating the isolation chamber ( 7 ), the isolated genetic material is moved into a plurality of reaction chambers ( 8.1, 8.2, 8.3, 8.4 ), genetic material is amplified by heating the reaction chambers ( 8.1, 8.2, 8.3, 8.4 ), lyophilized reagents for genetic material amplification together with lyophilized fluorescent tag intercalating with genetic material are present in the reaction chambers ( 8.1, 8.2, 8.3, 8.4 ), and signal detection from fluorescent tags is carried out along with the genetic material amplification stage.

The object of the invention is a method of detecting genetic material(including DNA and RNA) in a biological material sample, in particularusing LAMP technology (Loop-mediated Isothermal AMPlification) foramplifying genetic material and the device for its implementation. Theobject of the invention is used for rapid and mobile detection ofbacterial, viral and fungal pathogens in the biological materialobtained.

Currently there is a demand for rapid, inexpensive and effectivediagnostic methods to identify bacterial, viral and fungal pathogensthat may be microbial contaminants, e.g. of food products.

The US patent application US2009061450A1 discloses a device fordiagnosis and assay of respiratory pathogens, comprising a nasalsampling device, a single entry, disposable microfluidic cartridge fortarget nucleic acid amplification, and an instrument with on-board assaycontrol platform and target detection means. A device for sampling is asample carrier being placed in an appropriate receptacle in amicrofluidic cartridge so that they are sealingly and fluidly connectedto each other. A number of chambers may be distinguished in themicrofluidic cartridge, where subsequent steps of pathogen assay methodare performed, wherein first stage includes isolation of geneticmaterial from the tested sample, the isolated material is then amplifiedand subjected to detection. In one embodiment of cited solution,amplification of genetic material is accomplished using the LAMP method.In the reaction chamber, where amplification takes place, it isnecessary to provide a set and stable temperature, which in presentedsolution is achieved by the ITO heating element printed on amicrofluidic device. Fluorescent tag intercalating with genetic materialis added after performing genetic material amplification, allowingoptical detection in real time.

In turn, US patent application US2012264132A1 discloses a device andmethod of processing of samples, including essentially isothermalamplification of nucleic acids. The device according to a citedinvention comprises a first substrate having a first population areas,at least one area of the first population having at least one satellitearea disposed proximate to the at least one area, and at least onesatellite area being adapted to retain material from the first area. Thedevice additionally comprises a second substrate having a secondpopulation area formed therein, the first and second substrates beingengaged with one another such that the relative motion between the firstand second substrates places at least some of the first population areasin alignment with at least some of the second population areas so thatthey are in fluid communication with one another. Amplifying geneticmaterial using the mentioned device consisting in contacting a samplematerial disposed in a plurality of first areas, the sample materialcomprising a nucleic acid target, and at least one of the first areascontaining one molecule of the nucleic acid target, with a reactantmaterial disposed in a plurality of second areas, the contacting beingeffected by pairwise placement of at least some of the first areas andat least some of the second areas into direct fluid communication withone another. The said contacting of the materials effects amplificationof nucleic acid target molecule.

US patent application US20140335527A1 discloses a system and method formobile analysis of nucleic acids and proteins. Mobile analysis system isa small wireless device, which communicates with the used via thedisplay and keyboard. Mobile analysis system is using connected modulesfor extracting, amplifying and detecting nucleic acids from the samples.The entire process, together with data processing takes usually not morethan 20 minutes. In the first stage of the analysis method of geneticmaterial the biological sample is loaded onto an integrated chip. Theloading of the biological sample can be accomplished manually, throughsample inlet port or through an automated sampling. In the integratedchip the sample is transported to an extraction module in which theprocess of extracting genetic material from the biological sample isperformed. The isolated nucleic acids are then transported to theamplification module, in which in one embodiment amplification isperformed using LAMP method. The extraction and amplification methodscontain all the reagents needed to carry them out. Amplificationrequires retaining set increased temperature, which can be achievedthrough infra-red heating elements. Amplified genetic material goes tothe detection module in which it is detected, for example, by afluorescent signal derived from appropriate tags attached to thedetected DNA. Therefore, one of the chambers of genetic materialamplification may be preloaded with e.g. fluorescently tagged LAMPmaster mix. The entire integrated chip is transparent, allowingtransmission of light beams for heating the respective modules anddetecting fluorescence signal.

A technical issue to be solved is providing a method of detectinggenetic material (in particular DNA and/or RNA) in a biological materialsample and the device for its implementation which will allow rapiddetection of preferred pathogens, at the same time, the device will besimple to build, complete, mobile, relatively inexpensive to manufactureand will allow storage of reaction cartridges for extended periods oftime and will not be associated with specific storage conditions such asvery low temperatures. It is also preferred for the reaction cartridges,being a part of the device for detecting the pathogen in the biologicalmaterial sample, to be suitable for disposal and the device itself tohave limited energy consumption. Moreover, it is preferred that thedeveloped method of pathogen detection reduces the number of stepsrequired, making it simpler and faster to implement and that theconstruction of the device for its implementation provides a reducedrisk of contamination of the biological material sample. Surprisingly,the above-mentioned issues have been solved by the invention shown.

First object of the invention is a method of detecting genetic materialin a biological sample including the following stages:

a) the biological sample is loaded into the reaction cartridge and thenor before that reaction cartridge is placed in the measurement device,b) the collected biological sample is taken to the isolation chamber,c) isolation of biological material from the tested sample by heatingthe isolation chamber,d) the isolated genetic material is moved into a plurality of reactionchambers,e) genetic material is amplified by heating the reaction chambers,characterized in that inside at least one of reaction chambers arepresent freeze-dried reagents for amplification of genetic materialtogether with luminescent dye, comprising fluorescence dye orquantum-dots binding genetic material to be detected, whereassimultaneously with the stage of amplification of genetic material adetection of luminescent signal from luminescent markers is registered

In preferred embodiment of the invention a biological sample is takenfrom a sampling system and stage a) is performed by loading the samplingsystem into to the reaction cartridge (6).

In another preferred embodiment of the invention heating of theisolation chamber and/or reaction chamber is performed through aplurality of heating units on LEDs with temperature detectors,preferably emitting electromagnetic radiation with a wavelength in therange of 350 nm to 530 nm.

In another preferred embodiment of the invention the heating unit ofLEDs with temperature detectors comprises an optical temperaturedetector that detects electromagnetic radiation in the wavelength rangeof 4 μm to 12 μm.

In another preferred embodiment of the invention a biological sample istaken using capillary forces for the capillary in the sampling system.

Preferably lyophilized reagents for genetic material amplificationinclude deoxynucleotides, specific primer sequences, reaction buffer,magnesium ions Mg²⁺, preferably in the form of MgSO₄, polymerase capableof carrying out an amplification reaction, preferably Bst 3.0polymerase.

Equally preferably, lyophilized fluorescent tag intercalating withdetected genetic material is SYBR Green.

For detection, according to the first and second aspect of theinvention, the real-time detection of nucleic acid amplification productas well as the end-point technique oligonucleotides with a quantum dotmolecule attached at the 5′ end and a quencher attached at the 3′ end.The sequence of the oligonucleotides used is complementary to theportion of the amplified region of the deoxyribonucleic acid fragmentlocated between the designed primers F1 and B1 c and for the portion ofthe amplified region of the nucleic acid fragment located between thedesigned primers F1 c and B1. During the amplification reaction, apolymerase having a strand displacement activity and 5′>3′ exonucleaseactivity is used, e.g. Bst DNA Polymerase, Full Length.

During the deoxyribonucleic acid amplification reaction, the probe bindsto the complementary fragment in the amplified DNA segment, during theamplicon elongation, due to the exonuclease properties of thepolymerase, the attached oligonucleotide is degraded which results inseparation of the quencher from the quantum dot. As a result ofseparation, the quencher from the quantum dot, electromagnetic radiationis emitted in the UV, IR or VIS range after excitation of the quantumdot with the radiation wave-length specific for the material from whichthe quantum dot was created. The emitted signal is registered by thephotosensitive element.

The use of quantum dots causes a significant reduction in the detectionthreshold due to the possibility of using a source of excitation lightwith a higher power, thanks to which it is possible to register theemission of electromagnetic radiation coming from a much smaller amountof released quantum dots. In addition, the use of quantum dots formarking oligonucleotide fragments allows for a better separation ofexcitation wavelength from the wave-length of emission signal in whichdetection of electromagnetic radiation occurs. What is more, quantumdots have an increased bleaching durability compared to traditionalfluorochromes, which facilitates detection throughout the entireamplification reaction

More preferably, the reaction cartridge comprises three reactionchambers, including a test chamber including specific primers for thegenetic material tested, a positive control chamber that containsprimers specific to a particular portion of the genetic material fromwhich the biological material sample is derived and a negative controlchamber, containing reaction components without primers.

In preferred embodiment of the invention the reaction chambers in a topview are circles, complementary and interconnected in the middle with avalve or a diaphragm.

In another preferred embodiment of the invention at stage c) theisolation chamber is heated to 95° C. from 5 minutes to 10 minutes.

In yet another preferred embodiment of the invention at stage e) thereaction chambers are heated to 65° C. from 15 minutes to 60 minutes.

Preferably stage b) is accomplished by means of a first pump, preferablyin the form of a water tank closed with a diaphragm connected to apressure-producing chamber or piston and bellows.

Equally preferably, step d) is accomplished by means of a second pump,preferably in the form of a hollow chamber closed with a diaphragmconnected to a pressure-producing chamber.

More preferably, the method additionally comprises a stage of heating areaction cartridge to to temperatures above 100° C., preferably througha number of heating units on LEDs with temperature detectors.

Second object of the invention is a device for detecting geneticmaterial in a biological sample comprising a reaction cartridge andmeasurement device, the measurement device comprising a measurementchamber having a receptacle housing the reaction cartridge, wherein thereaction cartridge comprises an isolation chamber for isolating geneticmaterial, which is connected with the reactions chambers through thechannels, for amplifying isolated genetic material, characterized inthat inside at least one of reaction chambers are present freeze-driedreagents for amplification of genetic material together with luminescentdye, comprising fluorescence dye or quantum-dots binding geneticmaterial to be detected, whereas simultaneously with the stage ofamplification of genetic material a detection of luminescent signal fromluminescent markers is registered

In preferred embodiment of the invention the device comprises adetachable sampling system, the detachable sampling system comprising aplug and the reaction cartridge comprising a receptacle fitted to saidplug and providing a stable and tight fluid connection between thesampling system and the reaction cartridge.

In another preferred embodiment of the invention the device additionallycomprises a measurement module for image control and analysis,communication module, power supply module and display module.

In another preferred embodiment of the invention the measurement devicecomprises a plurality of heating units on LEDs with temperaturedetectors, preferably emitting electromagnetic radiation with awavelength in the range of 350 nm to 530 nm, arranged substantiallyopposite the isolation chamber and reaction chambers such that the lightbeams emitted by said plurality of LEDs illuminate said isolationchamber and reaction chambers.

In another preferred embodiment of the invention the heating unit ofLEDs with temperature detectors comprises an optical temperaturedetector that detects electromagnetic radiation in the wavelength rangeof 4 μm to 12 μm.

Preferably, the reaction chambers in a top view are circles,complementary and interconnected in the middle with a valve or adiaphragm.

Equally preferably, the sampling system comprises a capillary, to whicha biological sample is taken, connected with a first pump, preferably inthe form of a water tank closed with a diaphragm connected to apressure-producing chamber or piston and bellows.

More preferably, lyophilized reagents for genetic material amplificationdeoxynucleotides, specific primer sequences, reaction buffer, magnesiumions Mg²⁺, preferably in the form of MgSO₄, polymerase capable ofcarrying out an amplification reaction, preferably Bst 3.0 polymerase.

In preferred embodiment of the invention, lyophilized fluorescent agentintercalating with detected genetic material is SYBR Green.

In yet another preferred embodiment of the invention, the reactioncartridge comprises three reaction chambers, including a test chamberincluding specific primers for the genetic material tested, a positivecontrol chamber that contains primers specific to a particular portionof the genetic material from which the biological material sample isderived and a negative control chamber, containing reaction componentswithout primers.

In yet another preferred embodiment of the invention, the reactioncartridge comprises a second pumps, preferably in the form of an emptychamber closed with a diaphragm connected to a pressure-producingchamber, causing the movement of isolated genetic material from theisolation chamber to the reaction chambers.

Preferably, the reaction cartridge and/or sampling systems is made of ahydrophobic polymer and is a fully passive system.

Equally preferably, the isolation chamber and reaction chamber as wellas the second pump in the reaction cartridge comprise the valves,preferably optical ones on the inlet and outlet channels, respectively.

More preferably, in the channel connecting the isolation chamber withthe second pump there is a liquid detector, preferably a reflectiveinfra-red one.

In preferred embodiment of the invention, liquid detectors, preferablyreflective infra-red ones are located in outlet channels from thereaction chambers.

In another preferred embodiment of the invention, the measurementchamber has a controlled isothermal temperature in the range from 4° C.to 40° C., realized via a heating system, preferably in the form of aPeltier assembly.

In another preferred embodiment of the invention, the heating systemcomprises a connected fan and radiator, and an air-mixing wheel islocated in the measurement chamber.

Preferably, the measurement chamber is insulated with thermalinsulation.

Equally preferably, the device comprises a positioning mechanism of thereaction cartridge.

More preferably, the device comprises a pressure setting mechanismexerting a pressure on the pump in the reaction cartridge and anoppositely set pressure sensor.

In preferred embodiment of the invention, the device comprisesadditional UV LEDs illuminating the detection area.

In another preferred embodiment of the invention, the device comprisesadditional LEDs for operating liquid detectors and valves.

In another preferred embodiment of the invention, at the bottom of theisolation chamber and/or reaction chamber there is an absorption layerabsorbing photon energy, preferably made of Cu or Al coated with oxidespreferably, dyed black Al₂O₃.

A method of detecting genetic material in a biological sample accordingto the present invention allows to avoid the need to modify thebiological material sample by placing it in the devices, reducing theprobability of contamination and also allows the user to perform thetest only by the end user. Moreover, no additional laboratory equipmentor sterile reaction preparation conditions are required to complete thetest. Additionally, lyophilization in the production process of thereaction components provides a significant increase in the usefulness ofthe reaction cartridge (even more than one year from the date ofmanufacture), and it is not necessary to store the reaction cartridgeunder refrigeration. Placing the primers, specific for the amplifiednucleic acid fragment, inside the reaction chamber additionally reducesthe susceptibility of the procedure to contamination, and furtherfacilitates the study to the end user. In addition, placement of the dyein the reaction chamber enables immediate detection of the resultingreaction product without the end user taking action and significantlysimplifies the entire detection process by reducing the number of stepsrequired. The reaction cartridge, as well as the sampling system, aremade, as fully passive components, from one polymer material, allowingthem to be safely disposed of, benefiting the environment. In addition,LEDs for heating the isolation chamber and the reaction chambers used inthe control device reduce the energy consumption of the whole process.

Exemplary embodiments of the invention are shown in figures of thedrawing, in which FIG. 1, 6 shows a schematic representation of thesampling system and the reaction cartridge according to one embodimentof the present invention, FIG. 2, 7 shows the reaction cartridgeaccording to another embodiment of the present invention, FIG. 3 showsthe reaction cartridge according to yet another embodiment of thepresent invention, FIG. 4 shows various embodiments of the valves usedin different embodiments of the reaction cartridge, while FIG. 5 shows ablock diagram of the measurement device according to one embodiment ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic representation of the sampling system and thereaction cartridge according to one embodiment of the present invention.

FIG. 2 shows the reaction cartridge according to another embodiment ofthe present invention.

FIG. 3 shows the reaction cartridge according to yet another embodimentof the present invention.

FIG. 4 shows various embodiments of the valves used in differentembodiments of the reaction cartridge.

FIG. 5 shows a block diagram of the measurement device according to oneembodiment of the present invention.

FIG. 6 shows a schematic representation of the sampling system and thereaction cartridge according to one embodiment of the present invention.

FIG. 7 shows the reaction cartridge according to another embodiment ofthe present invention.

EXAMPLE 1

A device for detecting genetic material in a biological sample accordingto embodiment of the present invention was partially illustratedschematically (without a measurement device) in FIG. 1 and its varietyon FIG. 6. The biological material sample (e.g. capillary blood, wholeblood, saliva, body cavity fluid) taken using the sampling system 1, isintroduced into a reaction cartridge 6 made from a hydrophilic polymercoated with an anticoagulant layer, e.g. sodium citrate, EDTA, bycapillary forces in a volume not exceeding 1 ml (in the embodiment shownin FIG. 1 the volume is 10 μl). After filling the capillary 2, signaledto the end user by means of a tag at the end of the channel (not shown),for example by observing the channel filling up to the control window orby

means of a light and/or sound signal, the biological material istransported by the pressure of the fluid exiting the chamber containingwater intended for molecular diagnostics.

The mixture of biological material and water passes from the capillary 2into the isolation chamber 7 at its end. In the isolation chamber 7there is an Chelex 100 immobilized ion exchange resin or other materialcapable of binding inhibitors of the amplification reaction of geneticmaterial. After the mixture has passed through, the contents of theisolation chamber 7 is heated to 70° C. or higher for more than 5minutes. At this time, there is a thermal lysis of the cells and, insome cases, also the viral nucleocapsides contained in the biologicalmaterial, thus releasing the genetic material from their inside.Depending on the type of pathogen, the temperature can be increased to98° C. At the end of the heating process, the mixture is cooled(passively or actively, e.g. by stream of air flowing through the fan)and moved to at least one reaction chamber 8.1, 8.2, 8.3, 8.4 at avolume of at least 0.1 μl (in the embodiment shown in FIGS. 1 and 6reaction chambers 8.1, 8.2, 8.3, 8.4 have a volume of 20 μl) in which alyophilisate, containing the appropriate amounts of substances necessaryto perform a specific isothermal amplification reaction and detection ofselected, fragment of genetic material amplified during the reaction, islocated. Lyophilization in the production process of the reactioncomponents allows a significant increase in the usefulness of thereaction cartridge (even more than one year from the date ofmanufacture), and it is not necessary to store the reaction cartridgeunder refrigeration. The reaction chamber 8.1, 8.2, 8.3, 8.4 houses alsoa lyophilized dye intercalating with DNA. Placement of the dye in thereaction chamber enables immediate detection of the resulting reactionproduct without the end user taking action. Depending on the fluorescentdye used, the length of the light that causes the intercalating dye isdifferent. Dyes used for marking give visible light. The fluorescent dyeintercalating with DNA (for example, SYBR Green, EvaGreen, PikoGreen,Ethidium bromide, Calcein, Acridine Orange, Proflavin, Acriflavine andothers) is used to detect the amplification reaction product.

For detection, according to the first and second aspect of theinvention, the real-time detection of nucleic acid amplification productas well as the end-point technique oligonucleotides with a quantum dotmolecule attached at the 5′ end and a quencher attached at the 3′ end.The sequence of the oligonucleotides used is complementary to theportion of the amplified region of the deoxyribonucleic acid fragmentlocated between the designed primers F1 and B1 c and for the portion ofthe amplified region of the nucleic acid fragment located between thedesigned primers F1 c and B1. During the amplification reaction, apolymerase having a strand displacement activity and 5′>3′ exonucleaseactivity is used, e.g. Bst DNA Polymerase, Full Length.

During the deoxyribonucleic acid amplification reaction, the probe bindsto the complementary fragment in the amplified DNA segment, during theamplicon elongation, due to the exonuclease properties of thepolymerase, the attached oligonucleotide is degraded which results inseparation of the quencher from the quantum dot. As a result ofseparation, the quencher from the quantum dot, electromagnetic radiationis emitted in the UV, IR or VIS range after excitation of the quantumdot with the radiation wave-length specific for the material from whichthe quantum dot was created. The emitted signal is registered by thephotosensitive element.

The use of quantum dots causes a significant reduction in the detectionthreshold due to the possibility of using a source of excitation lightwith a higher power, thanks to which it is possible to register theemission of electromagnetic radiation coming from a much smaller amountof released quantum dots. In addition, the use of quantum dots formarking oligonucleotide fragments allows for a better separation ofexcitation wavelength from the wave-length of emission signal in whichdetection of electromagnetic radiation occurs. What is more, quantumdots have an increased bleaching durability compared to traditionalfluorochromes, which facilitates detection throughout the entireamplification reaction

In order to amplify the specific reaction product, the followingisothermal amplification technologies may be used: Loop-mediatedisothermal amplification (LAMP); Strand displacement amplification(SDA); Helicase-dependent amplification (HDA); Nicking enzymeamplification reaction (NEAR). Lyophilisate contains the experimentalamounts of deoxynucleotides (dNTPs); specific primer sequences, reactionbuffer components, magnesium ions Mg²⁺; polymerase capable of carryingout an amplification reaction; in some cases, reverse transcriptase andother components necessary to amplify the selected sequence of geneticmaterial. A set of primers (at least a pair of primers) with a uniquesequence specific for the genome of a given pathogen determines thespecificity of the reaction. The water coming from the isolation chamber7 together with the material dissolved therein is loaded into thereaction chamber 8.1, 8.2, 8.3, 8.4.

The amplification process of a selected nucleic acid fragment takesplace in the reaction chamber 8.1, 8.2, 8.3, 8.4 at a constanttemperature of at least 40° C. for a minimum of 5 minutes. The specificprimer sequences are binding to the template DNA (isolated in thepre-isolation chamber 7), derived from the various pathogens present inthe biological material. If the biological material is RNA, theamplification process is preceded by reverse transcription using theso-called random primers, resulting in cDNA. Once specificity has beendetermined by the primer, the DNA polymerase synthesizes thecomplementary strand. During the LAMP process about 30 μg/μl of DNA isreceived. Such a large amount of double-stranded DNA is shown by thedyes intercalating with the genetic material. By adding a fluorescentdye to the lyophilisate, the combining of the dye with the DNA occurssimultaneously with its amplification during the reaction. Uponcompletion of the reaction, the reaction mixture is illuminated with alight of a specific wavelength which excites the dye intercalating withDNA on a fluorescence basis. Detection of the reaction product isachieved by registering, the wavelength emitted by the dye anddouble-stranded DNA complex specific for the dye used, using thephotoconductor unit. The construction of the reaction cartridge 6 andthe material from which it was made (i.e. transparent polymer), allowsthe transmission of the light both exciting the dye-DNA complex as wellas the light emitted by this complex. The result is interpreted on thebasis of the presence of light or its absence (positive result—currentlight, negative—no light).

EXAMPLE 2

In the present embodiment the device for detecting the genetic materialin the biological sample comprises in general three main elements, i.e.measurement device (shown in the form of a block diagram in FIG. 5), thereaction cartridge 6 and the sampling system 1. FIG. 1 schematicallyshows a construction of one, non-limiting embodiment of the reactioncartridge 6 and sampling system 1. Connected sampling system 1 and thereaction chamber 6 are arranged in the measurement device which isdesigned to steer and control the whole process of genetic materialanalysis. The measurement device takes the form of a small mobiledevice, like a mobile phone, which contains a receptacle housing thereaction cartridge 6.

The sampling system 1 comprises a blood-collecting capillary 2 which isconnected to the water chamber, which is a water tank closed with adiaphragm connected to the pressure-producing chamber. Such a systemworks as the first pump 1 producing pressure exerting water from thewater chamber through a capillary 2 with a sampled biological material.Proper operation of the sampling system 1 provides the vent 3, whichforms the branching of the capillary 2 and jointly controlled valves Z1and Z2. During use, the sampling system 1 is in contact with liquidbiological material (e.g. blood), where the capillary 2 is filled underthe influence of capillary forces When filling the capillary 2 withbiological material, the valve Z1 is open and the valve Z2 is closed toensure proper operation of the system. After filling the capillary 2with the biological material, the sampling system 1 is placed in thereaction cartridge 6. A tight and stable connection of these elements isprovides by a matching plug 4 in the sampling system 1 and a receptacle5 in the reaction cartridge 6. The connection of this plug 4 to thereceptacle 5 provides a stable and sealed fluid connection between thesampling system 1 and the reaction cartridge 6. After placing thesampling system 1 in the reaction cartridge 6, the valve Z1 is closed,the valve Z2 is opened, and the activation of the first pump P1 (i.e.water tank closed with a diaphragm connected to the pressure-producingchamber). Activation of the first pump P1 occurs by mechanicalcompression of the chamber. The activation method of the first pump P1is not limiting in this case, and any method known in the prior art maybe used to transport of the liquid, e.g. heating with LEDs a substancewith a high thermal expansion coefficient. This operation removes thebiological material from the capillary 2 together water from the waterchamber. The mixture of water and biological material is transportedthrough a suitable channel to the isolation chamber 7. In the isolationchamber 7 there is a material capable of binding the inhibitors ofamplification reaction of the genetic material, and the isolationchamber 7 has access to the water chamber. Collected biological materialis provided into this isolation chamber 7. The capacity of thisisolation chamber is about 100 μl. There is a connecting channel with ahollow chamber closed with a diaphragm connected to a pressure-producingchamber forming a second pressure-generating pump P2, extending from theisolation chamber 7. The isolation chamber 7 is connected to the secondpump P2 by an reflective infra-red liquid detector D1 and a normallyopen valve Z3. The second P2 pump in turn is connected by a normallyopen valve Z4 with a vent 9 located at the end of the reaction cartridge6, opposite to the receptacle 5. This configuration of the valves Z3 andZ4 allows the mixture of biological material and water to be introducedthrough the isolation chamber 7 further towards the second pump P2. Whenthe test mixtures reaches the liquid detector D1, the isolation chamber7 signals its filling and the Z11 and Z3 valves are closed. Then, thebiological material in the isolation chamber 7 is heated to a suitabletemperature for a specified time period, which causes the release of thegenetic material encapsulated in the cells/protein envelope.

After the stage of isolating the genetic material from the collectedsample is completed, the valves Z3, Z5, Z6 and Z7 are opened and thesecond pump P2 is activated. The valves Z5, Z6 and Z7 are located onseparate channels connecting the isolation chamber 7 to thecorresponding reaction chambers 8.1, 8.2, 8.3, 8.4. Each reactionchamber 8.1, 8.2, 8.3, 8.4 in turn is connected with a correspondingvent 9 located on the edge of the reaction cartridge 6, via liquiddetectors D1, D2, D3, respectively, and normally open valves Z8, Z9,Z10, respectively. Activation of the second P2 pump, along with theconfiguration of the valves Z5, Z6, Z7 and Z8, Z9, Z10 allows theisolated genetic material to be moved into the reaction chambers 8.1,8.2, 8.3, 8.4. After receiving the signal from the D1, D2, D3 liquiddetectors the valves Z8, Z9, Z10 are closed. Then, the valves Z5, Z6 andZ7 are closed next. In this way, the reaction chambers 8.1, 8.2, 8.3,8.4 are filled with the isolated genetic material. The reaction chambers8.1, 8.2, 8.3, 8.4 contain lyophilized reagents in their volume,containing all the necessary ingredients for the amplification of thegenetic material. Master mix in the reaction chambers 8.1, 8.2, 8.3, 8.4comprises also a lyophilized fluorescent dye intercalating with geneticmaterial. The capacity of the reaction chambers 8.1, 8.2, 8.3, 8.4 is inthe range of 20 μl to 25 μl. In the present embodiment three reactionchambers 8.1, 8.2, 8.3, 8.4 are provided, including a test chamber 8.1comprising specific primers for the genetic material tested, a positivecontrol chamber 8.2 that contains primers specific to a particularportion of the genetic material from which the biological materialsample is derived and a negative control chamber 8.3 that does notcontain primers, but other reaction components. The positive controlchamber 8.2 is designed to allow for control of the polymerase,temperature conditions and the isolation of the genetic material. Thenegative control chamber 8.3 allows to control the lyophilizationprocess (e.g. sterility) and control of the valve behaviour, which couldcause mixing of the contents of these reaction chambers. Of course, thenumber of chambers used is not a limitation of the present invention,and the person skilled in the art will, for example, use a simultaneousincrease in the number of chambers 8.1, 8.2, 8.3, 8.4 for thesimultaneous analysis of different pathogens.

To amplify the genetic material, the reaction chambers 8.1, 8.2, 8.3,8.4 are then heated to the appropriate temperatures, which amplifies thegenetic material. Simultaneously with the amplification (orsubsequently) the fluorescence signal detected from the fluorescent tagused is attached to the amplified genetic material. Product increment isequal to the increase in light intensity generated by the fluorescenttag used.

After the whole process and reading the result by the optical systemwith the camera 28, the regions containing the biological material areheated with UV LEDs 29 emitting radiation at wavelengths ranging from350 nm to 450 nm (or laser) to 150° C. for 2 to 3 second to neutralizebiological hazard. At lower UV power, these UV LEDs 29 simultaneouslyserve to excite fluorescence (illuminate reaction cartridge 6). UVexposure results in the destruction of biological material anddepolymerization of the reaction chamber material 6, which reduces thebiological hazard and disintegrates the polymer, favourably protectingthe environment and ensuring proper disposal.

Throughout the process of biological material analysis, the thermaltreatment of the liquid biological material is carried out in theisolation chamber 7 and in the reaction chambers 8.1, 8.2, 8.3, 8.4. Theenergy required to heat the isolation chamber 7 and the reactionchambers 8.1, 8.2, 8.3, 8.4 is communicated without contact. The sourceof energy is the LED light emitting diodes, which emit light radiationfrom the UV-VIS range. For example, wavelengths emitted by LEDs can beselected from 350 nm to 500 nm. The light emitting diodes are locatedinside the measurement device and are arranged to illuminate the area ofthe isolation chamber 7 and the reaction chambers 8.1, 8.2, 8.3, 8.4. Byusing a transparent material for the construction of the reactioncartridge 6, which is characterized by high light transmission, it ispossible to use an energy-efficient heating method for the respectivechambers. The temperature of the reaction chamber 8.1, 8.2, 8.3, 8.4 andisolation chamber 7 is controlled with no contact by a pyrometer with adigital processing block. The entire system is controlled by amicroprocessor driver with built-in software. Furthermore, the low-powerUV LED is used in the measuring device to illuminate the inside of thereactor, which is necessary for image recording by the CCD. Detection ofthe biochemical reaction product is based on determining the quantizedlevels of signals from the CCD detector RGB channels. The design of thedevice allows for continuous recording of colour signals. Using theilluminating diode allows continuous recording of the image by thedetector, as it is not necessary to constantly illuminate the samplewith an external light source.

Construction of the measurement device according to one embodiment ofthe present invention is shown in block diagram form in FIG. 5. Ingeneral, a measurement chamber 10 which is constructed so that it housesa reaction cartridge 6 is distinguished in the measurement device. Inorder to properly position the reaction cartridge 6 in the measurementchamber 10, a positioning mechanism 11 of the reaction cartridge 6 isprovided. The measurement chamber 10 is a closed structure that iscovered by an outside thermal insulation 12, which facilitates keepingthe set temperature inside. Maintaining the isothermally controlledtemperature inside the measurement chamber 10 (e.g. in the range of 4°C. to 40° C.) ensures the heating system 13 (e.g. in the form of aPeltier assembly). In order to properly distribute the heated air insidethe measurement chamber 11, a fan 14 and an air-mixing wheel 22 areused. The efficiency of the heating system 13 is ensured also by theradiator 15 located outside the measurement chamber 10. The measurementdevice also contains further blocks necessary for the proper functioningof the device, such as the image control and analysis module 16, thecommunication module 17, the power supply module 18, and the display 19.The functionality of the above-mentioned blocks and their constructionare well known to those skilled in the art, so their exact descriptionis omitted to simplify the discussion. The measurement chamber 20 of themeasurement device provides also the pressure setting mechanism 10 forthe purpose of activating the pump P1 in the reaction cartridge 6. Apressure sensor 21 is located opposite to control the set pressure onpump P1. In order to ensure a correct temperature in the isolationchamber 7 and the reaction chambers 8.1, 8.2, 8.3, 8.4, a number ofheating units 23 are provided in the measurement chamber 10 which are sopositioned relative to the reaction cartridge 6 that the emitted lightstreams illuminate the isolation chamber 7 and the reaction chambers8.1, 8.2, 8.3, 8.4, respectively. More specifically, each heating unit23 consists of LEDs with radiators 24, a temperature detector 25, and atemperature detector stabilizer 26. Heating of the isolation chamber 7and the reaction chamber 8.1, 8.2, 8.3, 8.4 is performed with LEDs witha continuously adjustable photon energy stream ranging from 400 nm to500 nm, which is preferred due to high photon emission performance andtranslates into high power reaching the absorption layer at the bottomof the isolation chamber 7 and reactor chambers 8.1, 8.2, 8.3, 8.4. Inorder to compensate for the temperature of the bottom of the chamber 7,8.1, 8.2, 8.3, 8.4 and absorption of energy, to prevent degradation ofthe lyophilized biological material in chambers 8.1, 8.2, 8.3, 8.4., theabsorption layer completely absorbing photon energy was used. The layeris made of materials e.g. Cu or Al coated with oxides (in the presentexample Al₂O₃, dyed black) and other materials with good absorptionproperties and good thermal conductivity (including modified polymerse.g. carbon or graphene).

The temperature increase over time in chambers 7, 8.1, 8.2, 8.3, 8.4 isachieved by increasing the power of the light stream and decreasethrough the isothermal measurement chamber 10 at a temperature from 4°C. to 40° C. With the constant thermal resistance of the isolationchamber 7 or reactor chamber 8.1, 8.2, 8.3, 8.4 to the surroundings, therate of the decreasing temperature can be controlled by change theambient temperature of the reaction cartridge 6. Depending on thedesired temperature decrease rate, the temperature inside the device(i.e. in the measurement chamber 10) is set and a suitable power isapplied to the absorption layer of chambers 7, 8.1, 8.2, 8.3, 8.4. Inthis way any temperature profile can be obtained in the range from 25°C. to 100° C. with high increase and decrease rates. The absorptionlayer of the chambers has a high thermal conductivity which eliminatesthe possible heterogeneity of the light stream from the LEDs and ensuresno temperature gradients in the area of the working chambers.

Because the temperature measurement is done by a temperature detectorsuch as a pyrometer with a built-in radiation permeable filter in therange of 8 μm to 12 μm, it is possible to simultaneously measure thetemperature and supply energy to the isolation chambers 7 and reactionchambers 8.1, 8.2, 8.3, 8.4. In this case there are no periods of lackof control over the temperature control in the isolation chambers 7 andreaction chambers 8.1, 8.2, 8.3, 8.4. In addition, there are no peaks oftemperature associated with the operation of the PID controller, andthere is no need for power control by means of pulse-width modulationPWM, which has the advantage of producing less thermal noise.

In addition, a series of LEDs 27, analogically arranged so that the beamof light generated, illuminates the device, is provided to operate theZ1-Z11 valves and D1-D4 liquid detectors. The optical system with thecamera 28 which may have the form of a CCD detector and is intended todetect the light signal resulting from fluorescent dyes resulting fromreaction in the reaction chamber 8.1, 8.2, 8.3, 8.4. In order to allowthis UV LEDs 29 are also provided, which illuminate the detection area.

EXAMPLE 2

FIG. 2 schematically shows another embodiment of reaction cartridge 6used in the device for detecting genetic material in a biological sampleaccording to the present invention. The general design and principle ofoperation of the reaction cartridge 6 shown in the present embodiment isconsistent with the construction and principle of operation of thereaction cartridge 6 of Example 1. The fundamental difference betweenthe comparative reaction cartridges 6 is that in the reaction cartridge6 from example 2 integral sampling system 1 is used (it is not aseparate device as in the first embodiment of the present invention).The reaction cartridge 6 is therefore a compact structure, devoid ofdetachable elements. In this case, the collected biological material isintroduced into the sampling system 1, which forms an integral part ofthe reaction cartridge 6. Also in this example, the capillary 2 can bedistinguished, which by means of the capillary forces absorbs thebiological material. At the other end of the capillary 2 a controlwindow 30 is provided which signals the filling of the capillary 2 withthe biological material. In the present embodiment the first pump P1 ismade by means of mechanical elements such as piston and bellows. In thisembodiment It should be emphasized that water for the reaction chamber 6is provided in the form of capsules, which allows for easy sterilizationand the possibility of separating the wet process in the production ofthe reaction chambers 6. Water release takes place just before the testand is performed by needle injection 31 when the pump P1 startsoperating. Transport of the biological material and the products fromthe isolation chamber 7 to the reaction chambers 8.1, 8.2, 8.3, 8.4 isprovided by pump P1 by extruding water from the capsule. This simplifiesthe process control on the device. Suitable pressure during heating isprovided by the pressure sensor 21 in the measurement device and thecorresponding control of the valves Z5, Z8, Z9, Z10. In this embodiment,the construction of reaction chambers 8.1, 8.2, 8.3, 8.4 also deservesmentioning. Each of the reaction chambers 8.1, 8.2, 8.3, 8.4 in a topview are complementary circles. The reaction chambers 8.1, 8.2, 8.3, 8.4complement each other to form a circular region comprising all thereaction chambers 8.1, 8.2, 8.3, 8.4, connected in the middle by meansof a valve or diaphragm 33. In this embodiment, various valve designs 33may be used that do not affect the overallity of the embodiment.Exemplary valves 33, usable in reaction cartridges 6, are shown in FIG.4 A-D, hydrophobic circular valve, hydrophobic—mechanical round valve,hydrophobic—mechanical elliptic valve, hydrophobic—mechanicalrectangular valve, respectively. The presented mechanical valves act onthe deformation of the flexible material from which the valve was made.A specific force is required, which at the same time defines thepressure, which having been exceeded causes the liquid flow in a givendirection. The shape of the valve is such that in the second directionthe elastic deformation is blocked, thus blocking the flow of liquid forthat direction. The hydrophobic valves operate on the principle that theliquid must overcome the surface tension forces in contact with thehydrophobic valve material (while the air flows freely). This allows toblock the flow of the liquid in the channel to a predetermined pressuredepending on the diameter of the opening in the valve and thehydrophobicity of the material from which the valve is made. In the caseof simultaneous filling of the three channels with the liquid, afterplacing the hydrophobic valves at the ends of the channels they will beautomatically filled. The air will flow unobstructed, and the liquidwill stop successively on these valves, because more pressure will berequired to for the liquid to flow through the valves. The combinationof these two types of valves makes it easy to control the flow of liquidin the reaction cartridge 6.

Moreover, in the present embodiment no additional pump P2 is used andsubstantially the number of valves used was reduced (compared to thereaction cartridge 6 of the first embodiment). In addition, due to theconstruction of the reaction chambers 8.1, 8.2, 8.3, 8.4, the outletchannels are directed towards the three different edges of the reactioncartridge 6 and compensation chambers 33 are provided prior to the vent9 to prevent the liquid from exiting the reaction cartridge 6 into themeasurement device.

The other components and the principle of operation of the reactioncartridge 6 coincide with those disclosed in the first embodiment of thereaction cartridge 6.

EXAMPLE 3

The reaction cartridge 6 shown in FIG. 3, being yet another embodimentof the present invention, differs from the reaction cartridge 6 shown inFIG. 2 only in that in each reaction chamber 8.1, 8.2, 8.3, 8.4 twovalves 33 are used at the inlet and outlet of the reaction chamber 8.1,8.2, 8.3, 8.4) and a connecting channel 34 which is intended to providea stable and controlled liquid flow in one direction with pressurevariations caused by heating the reaction chambers 8.1, 8.2, 8.3, 8.4.In the solution presented in the present embodiment, in contract to thesolution presented in the second embodiment, there are no Z8 and Z10valves, which greatly simplifies the design of the reaction cartridge 6and its operation. The connecting channel 34 serves to vent the reactionchambers 8.1, 8.2, 8.3, 8.4. Due to the fact that the valves prevent theretraction of the liquid, there is no uncontrolled mixing of the liquidin the reaction chambers 8.1, 8.2, 8.3, 8.4. Placing two valves in thereaction chambers 8.1, 8.2, 8.3, 8.4 allows the use of only one valvenormally open at the outlet to ensure proper operation of the system.

EXAMPLE 4

Detection of HIV in the blood using the method of the present inventionand the device of the present invention.

To analyse the presence of HIV virus in a sample taken from a patient, amethod and device for the detection of genetic material in a biologicalsample according to the present

invention, described in detail in Examples 1 and 2. In isolation chamber7 Chelex 100 is used. DNA isolation involves the thermal degradation ofthe cell membrane or viral protein envelope and the release of geneticmaterial that is encapsulated in the viral cells/protein envelope.Chelex 100 is necessary to catch inhibitors that can block thepolymerase and produce false negative results. Chelex 100 is prepared asa 5% mixture in deionized water, nuclease-free, it can also beimmobilized at the bottom of the isolation chamber in the form of aporous layer. To perform isolation in the isolation chamber, the bloodis heated at 95° C. for 5-10 min.

Lyophilized reagents, including buffer, dNTPs, MgSO4, Primer Mixer, Bst3.0 polymerase, SYBR Green are in the reaction chambers. Theamplification of genetic material is carried out in reaction chambers8.1, 8.2, 8.3, 8.4 by heating at 65° C. for 30 min. There are specificHIV primers in the test chamber 8.1. In the endogenous positive controlchamber 8.2 there are specific primers for the human gene. In thenegative control chamber 8.3 there are no primers added, but is containsthe other components of the reaction. LAMP reaction and detection—takesplace in the reaction chambers 8.1, 8.2, 8.3, 8.4 and consists inamplifying genetic material of a given pathogen (and human geneticmaterial for endogenous control) using the Bst 3.0 polymerase enzyme.Specific primers added to the reaction are binding to selected fragmentsof the tested genome and determine the fragment amplified in thereaction. At the end of the reaction, approximately 10-50 μg/μl of theamplified DNA fragment is formed. SYBR Green present in the reactionmixture is combined with the reaction product and, when combined withdouble-stranded DNA, becomes fluorescent (illuminates when light is ofsufficient length). Product increment is equal to the increase in lightfrom the dye. At the end of the reaction, when the result is positiveand the tested fragment is amplified light is visible, when the resultis negative there is no light. Other reaction components (buffer, MgSO4,dNTPs) are added to provide suitable working conditions for Bst 3.0polymerase.

In the process of isolating the DNA/RNA material in the reactionchamber, the pathogen is neutralized. The only danger can be the residueof the genetic material in the capillary 2 or channels in the reactioncartridge 6. Thus, after the detection, the residue of the geneticmaterial is recycled, which is performed by exposing the reactioncartridge 6 (in particular the isolation chamber 7 and the reactionchambers 8.1, 8.2, 8.3, 8.4) to UV radiation to heat the individualcomponents to a temperature above 100° C. and thereby dispose of geneticmaterial. This allows to safely dispose of used reaction cartridge 6without having to carry out complicated disposal procedures.

1. A method of detecting genetic material in a biological sampleincluding the following stages: a) the biological sample is loaded intothe reaction cartridge (6) and then or before that reaction cartridge(6) is placed in the measurement device, b) the collected biologicalsample is taken to the isolation chamber (7), c) isolation of biologicalmaterial from the tested sample by heating the isolation chamber (7), d)the isolated genetic material is moved into a plurality of reactionchambers (8.1, 8.2, 8.3, 8.4), e) genetic material is amplified byheating the reaction chambers (8.1, 8.2, 8.3, 8.4), characterized inthat inside at least one of reaction chambers (8.1, 8.2, 8.3, 8.4) arepresent freeze-dried reagents for amplification of genetic materialtogether with fluorescence dye, comprising fluorescence dye orquantum-dots binding genetic material to be detected, whereassimultaneously with the stage of amplification of genetic material adetection of fluorescence signal from fluorescent markers is registered.2. A method according to claim 1 characterized in that a biologicalsample is taken from a sampling system (1) and stage a) is performed byloading the sampling system (1) into to the reaction cartridge (6)wherein heating of the isolation chamber (7) and/or reaction chamber(8.1, 8.2, 8.3, 8.4) is performed through a plurality of heating unitsof LEDs with temperature sensors (23), preferably emittingelectromagnetic radiation with a wavelength in the range of 350 nm to530 nm and the heating unit of LEDs with temperature detectors (23)comprises an optical temperature sensor (25) that detectselectromagnetic radiation in the wavelength range of 4 μm to 12 μm.
 3. Amethod according to any one of the claim 1, characterized in that abiological sample is taken using capillary forces for the capillary (2)in the sampling system (1) and lyophilized reagents for genetic materialamplification deoxynucleotides, specific primer sequences, reactionbuffer, magnesium ions Mg²⁺, preferably in the form of MgSO₄, polymerasecapable of carrying out an amplification reaction, preferably Bst 3.0polymerase wherein lyophilized fluorescent tag intercalating withdetected genetic material is SYBR Green.
 4. A method according to anyone of the claim 1, characterized in that the reaction cartridge (6)comprises four reaction chambers (8.1, 8.2, 8.3, 8.4), including a testchamber (8.1) including specific primers for the genetic materialtested, a positive control chamber (8.2) that contains primers specificto a particular portion of the genetic material from which thebiological material sample is derived and a negative control chamber(8.3), containing reaction components without primers.
 5. A methodaccording to claim 4, characterized in that the reaction chambers (8.1,8.2, 8.3, 8.4) in a top view are circles, complementary andinterconnected in the middle with a valve or a diaphragm (33).
 6. Amethod according to any one of the claim 1, characterized in that atstage c) the isolation chamber is heated to 95° C. from 5 min to 10 minwherein at stage e) the reaction chambers (8.1, 8.2, 8.3, 8.4) areheated to 65° C. from 15 minutes to 60 minutes and stage b) isaccomplished by means of a first pump (P1), preferably in the form of awater tank closed with a diaphragm connected to a pressure-producingchamber or piston and bellows and step d) is accomplished by means of asecond pump (P2), preferably in the form of a hollow chamber closed witha diaphragm connected to a pressure-producing chamber and comprises astage of heating a reaction cartridge (6) to temperatures above 100° C.,preferably through a number of heating units of LEDs with temperaturedetectors (23).
 7. A device for detecting genetic material in abiological sample comprising a reaction cartridge (6) and measurementdevice, the measurement device comprising a measurement chamber (10)having a receptacle housing the reaction cartridge (6), wherein thereaction cartridge (6) comprises an isolation chamber (7) for isolatinggenetic material, which is connected with the reactions chambers throughthe channels (8.1, 8.2, 8.3, 8.4), for amplifying isolated geneticmaterial, characterized in that inside at least one of reaction chambers(8.1, 8.2, 8.3, 8.4,) are present freeze-dried reagents foramplification of genetic material together with luminescent dye,comprising fluorescence dye or quantum-dots binding genetic material tobe detected, whereas simultaneously with the stage of amplification ofgenetic material a detection of luminescent signal from luminescentmarkers is registered.
 8. A device according to claim 7, characterizedin that the device comprises a detachable sampling system (1),comprising a plug (4) and the reaction cartridge (6) comprising areceptacle (5) fitted to said plug (4) and providing a stable and tightfluid connection between the sampling system (1) and the reactioncartridge (6) and a measurement module for image control and analysis(16), communication module (17), power supply module (18) and displaymodule (19).
 9. A device according to claim 7, characterized in that themeasurement device comprises a plurality of heating units of LEDs withtemperature detectors (23), preferably emitting electromagneticradiation with a wavelength in the range of 350 nm to 530 nm, arrangedsubstantially opposite the isolation chamber (7) and reaction chambers(8.1, 8.2, 8.3, 8.4) such that the light beams emitted by said pluralityof LEDs illuminate said isolation chamber (7) and reaction chambers(8.1, 8.2, 8.3, 8.4) wherein the heating unit of LEDs with temperaturesensors comprises an optical temperature detector that measureselectromagnetic radiation in the wavelength range of 4 μm to 12 μm andreaction chambers 18.1, 8.2, 8.3, 8.4) in a top view are circles,complementary and interconnected in the middle with a valve or adiaphragm.
 10. A device according to claim 7, characterized in that thesampling system (1) comprises a capillary (2) to which a biologicalsample is taken, connected with a first pump (P1), preferably in theform of a water tank closed with a diaphragm connected to apressure-producing chamber or piston and bellows and lyophilizedreagents for genetic material amplification deoxynucleotides, specificprimer sequences, reaction buffer, magnesium ions Mg²⁺, preferably inthe form of MgSO₄, polymerase capable of carrying out an amplificationreaction, preferably Bst 3.0 polymerase, wherein lyophilized fluorescenttag intercalating with detected genetic material is SYBR Green.
 11. Adevice according to claim 7, characterized in that the reactioncartridge (6) comprises four reaction chambers (8.1, 8.2, 8.3, 8.4),including a test chamber (8.1) including specific primers for thegenetic material tested, a positive control chamber (8.2) that containsprimers specific to a particular portion of the genetic material fromwhich the biological material sample is derived and a negative controlchamber (8.3), containing reaction components without primers, whereinthe reaction cartridge (6) comprises a second pump (P2), preferably inthe form of an empty chamber closed with a diaphragm connected to apressure-producing chamber, connected to the isolation chamber (7) andproducing pressure causing the movement of isolated genetic materialfrom the isolation chamber (7) to the reaction chambers (8.1, 8.2, 8.3).12. A device according to claim 7, characterized in that the reactioncartridge (6) and/or sampling systems (1) is made of a hydrophobicpolymer and is a fully passive system, wherein isolation chamber (7) andreaction chambers (8.1, 8.2, 8.3, 8.4) as well as the second pump (P2)in the reaction cartridge (6) comprise the valves (Z11), (Z5, Z6, Z7),(Z8, Z9, Z10), (Z3), (Z4), preferably optical ones on the inlet andoutlet channels, respectively.
 13. A device according to claim 7,characterized in that in the channel connecting the isolation chamber(7) with the second pump (P2) there is a liquid detector (D1),preferably a reflective infra-red one, wherein liquid detectors (D2, D3,D4), preferably reflective infra-red ones are located in outlet channelsfrom the reaction chambers (8.1, 8.2, 8.3, 8.4) and measurement chamber(10) has a controlled isothermal temperature in the range from 4° C. to40° C., realized via a heating system (13), preferably in the form of aPeltier assembly and the heating system (13) comprises a connected fan(14) and radiator (15), and an air-mixing wheel (22) is located in themeasurement chamber (10).
 14. A device according to claim 7,characterized in that the measurement chamber (10) is insulated withthermal insulation (12) and a positioning mechanism (11) of the reactioncartridge (6) and a pressure setting mechanism (20) exerting a pressureon the pump (P1) in the reaction cartridge (6) and an oppositely setpressure sensor (21) and additional UV LEDs (29) illuminating thedetection area.
 15. A device according to claim 7, characterized in thatcomprises additional LEDs (27) for operating liquid detectors (D1-D4)and valves (Z1-Z11) and at the bottom of the isolation chamber (7)and/or reaction chamber (8.1, 8.2, 8.3, 8.4) there is an absorptionlayer absorbing photon energy, preferably made of Cu or Al coated withoxides preferably, dyed black Al₂O₃.